The present invention relates to luminescent playthings which can be used preferably as candles, table lights, pen lights, illuminations, illuminating playthings for camps or night fishings, fish-gathering lamps, safety candles, neon lights, luminescent inks, luminescent pens, luminescent coatings or luminescent writing implements, in which a soft light with indescribable color tone not obtainable from flames of conventional candles is emitted via (or by adding) seawater, lake and marsh water, river water, ground water (including well water and hot-spring water), tap water, mineral drinking water, and the like (which are, in the present invention, referred to hereinafter as environmental water) by utilizing 3 components of luciferin, luciferase and ATP, or 4 components of these components plus a metal salt, out of components necessary for bioluminescence in fireflies or the like.
Conventionally, a table is decorated with candles in Christmas Eve or a birthday party, or in a wedding reception a candlelight service is conducted where the bride and groom light a candle in each table for invited guests, or in a coffee bar, people drink tea and have a meal in a table decorated with candles under a slightly different atmosphere in dark illumination in a coffee bar or the like. Under soft lights of these candles, foods and human faces look beautiful and the atmosphere becomes more enjoyable.
The object of the present invention is to provide a luminescent plaything for emitting a soft light with indescribable color tone not obtainable from conventional candles, to an ancillary material thereof, and to a method of storing a luminescent composition used therein.
The present inventors have now surprisingly found that the combination of luciferin, luciferase and ATP, substantially free of a metal salt, does not emit light even if dissolved in water such as distilled water (or pure water) or deionized water substantially free of a metal salt, but when dissolved in environmental water such as seawater and river water, it emits a soft light with indescribable color tone not obtainable from flames of conventional candles, presumably due to its reaction with a trace of a metal salt dissolved in such water, and luminescence utilizable as playthings can thereby be expected.
The present inventors have further found that a luminescent plaything comprising the combination consisting of 4 components of luciferin, luciferase, ATP and a metal salt, which emits light with a color tone not obtainable from conventional candles when these 4 components are divided into at least 2 groups and mixed so as to be contained simultaneously in the form of a liquid state in an objective place, and also that when a fifth component being a buffer is combined with the 4 components, the luminescence is sustained.
Still further, the present inventors have found that the concentrations of luciferin, luciferase, ATP and a metal salt can be selected from a wide range from low to high concentrations, and if their concentrations are low, luminescent playthings with commodity worthy cannot be obtained, that is, it cannot be expected that the luminescence is sufficiently satisfactory when observed with the naked eye and is worthy of appreciation, but if the 4 components of luciferin, luciferase, ATP and a metal salt are contained at concentrations of 0.01 mM or more, 0.015 mg/ml or more, 0.002 mM or more, and 0.02 mM or more respectively, it can be expected that the luminescence is sufficiently satisfactory when observed with the naked eye and is worthy of appreciation.
Further, the luminescent plaything might have the problem that it is poor in luminescent stability, and use of a large amount of ATP, luciferin and luciferase is inevitable to attain the long duration of luminescence.
As a result of the intensive study under these present circumstances, the present inventors have found that upon luminescence reaction proceeding by the action of pyruvate orthophosphate dikinase in the presence of phosphoenolpyruvic acid, pyrophosphoric acid, magnesium ions, luciferin, luciferase and ATP, the ATP consumed by the reaction is continuously regenerated, thereby providing a luminescent plaything with highly stable luminescence without increasing the amount of ATP, luciferin and luciferase added.
The shape of the luminous body of this luminescent plaything is subject to the shape of a container for accommodating the luminescent plaything, so there is the inconvenience that it cannot be enjoyed without considering the shape of the container. This can be solved by providing a container (glass container) of desired size and shape to meet various expected requests, but even if it is theoretically possible to provide a container of arbitrary shape, there is actually a limit to manufacturing techniques or costs, so there is a problem that it is not practical. Further, a luminescent plaything in the form of a solution or powder has the problem that the duration of its luminescence is poor.
As a result of the intensive study under these circumstances, the present inventors have found that in a luminescent plaything comprising the combination of 4 components of luciferin, luciferase, ATP and a metal salt, the 4 components being not simultaneously mixed in a liquid state, at least one of the 4 components is granulated so that beautiful luminescence of arbitrary shapes (e.g., circle-, star-, heart-, ball-, triangle-, and alphabet-shapes) is obtained in transparent water and the luminescence is sustained.
Further, the present inventors have found that the dissolution rate of the luminescent plaything in water can be arbitrarily controlled by changing the hardness of the granulated material and the bulk density of the texture, and thus the duration of luminescence can be controlled, and further the granulated material is structured in such a concentric circle that luminescent layers and non-luminescent layers are laminated alternately to form a multi-layer construction whereby the luminescence can be intermittently depressed as the granulated material is dissolved, or the shape and size of the granulated material are selected and its surface is sprayed with water or is brought into contact with a wet cloth whereby new types of luminescent playthings not known so far can be provided. Furthermore, luminescence generally utilizing a luciferase derived from an organism emits monochromatic (yellow or yellowish green) light, but this time, the present inventors developed luciferases emitting red light and orange light respectively, in addition to yellow light in the prior art. However, it is difficult that these 3 kinds of luciferases emit yellow, red and orange lights respectively in the same solution, and it is also difficult that these lights are enjoyed (because the lights are mixed), so it is strongly desired to solve this problem. The present inventors have found that the 3 lights (i.e., yellow, red and orange lights) are emitted in the same solution like a mirror ball by previously granulating these 3 kinds of luciferases and then mixing the granulated materials with other components necessary for luminescence.
Further, the resulting luminescent plaything in the form of a solution is mixed as such, or in the form of powder, is dissolved in water etc. in order to promote luminescence, but its luminous body does not have a shape, so there is the disadvantage that its luminescence cannot be enjoyed unless it is placed in a container. By making its granulated material, luminescence having a shape can be enjoyed, but as the granulated material is dissolved, the shape inconveniently collapses and disappears. Thus the granulated material cannot maintain luminescence having a specific shape, namely, luminescence having a three-dimensional structure.
As a result of the intensive study under these circumstances, the present inventors have found that of components necessary for bioluminescence, at least one or all components are adsorbed into, or allowed to adhere to, an arbitrary carrier, and then brought into contact with the remaining components in water or via water, whereby luminescence having a desired shape and a three-dimensional structure can be maintained not only inside the container but also outside the container, and luminescent playthings with good luminescence stability can thereby be obtained.
A mixture of powdery components necessary for bioluminescence, obtained in the present invention, or granules produced by granulating the mixture in a granulator, or a material produced by permitting the mixture to be adsorbed into or adhered to an arbitrary carrier and then drying it is/are readily eluted (or diffused) in water significantly, thus making topical luminescence difficult and shortening the luminescence time when it is used as luminescence bait for fishing.
As a result of the intensive study under these circumstances, the present inventors have found that when, of components necessary for bioluminescence, at least one or all components after optionally granulated or after adsorbed into or allowed to adhere to an arbitrary carrier are coated on the surface thereof with a water-soluble substance, the elution of the components necessary for bioluminescence into water is slightly delayed or the spreading of the components is prevented, thereby being capable of providing luminescent playthings in which the time to initiate luminescence can be controlled and topically strong luminescence is sustainable.
Further, this luminescent plaything in the form of a solution is mixed as such, or in the form of powder, is dissolved in water or the like in order to promote luminescence, but because its luminescence is emitted through the water surface or the wall (transparent window) of a transparent container, there is the problem that the luminescence remains no change thus being not enjoyable.
As a result of the intensive study under these circumstances, the present inventors have found that when water-insoluble materials such as glass marbles, rock ice (e.g., rock-shaped ice-like plastic or acrylic materials), novel-metal foil (e.g., aluminum foil) etc. are provided within the bioluminescent liquid, the luminescence is emitted irregularly thereby increasing the glitter and further increasing the beauty.
Further, this luminescent plaything emits a soft light with indescribable color tone not obtainable from flames of conventional candles, and the color tone of the luminescence depends on luminescence emitted by luciferase, so luminescence having a desired color tone is not obtainable inconveniently. Although only the method disclosed in Japanese Patent No. 2,666,561 is known as a method of changing a color tone for luminescence, luminescence having desired and fine tones of color is hardly obtainable even using this prior art method.
As a result of the intensive study under these circumstances, the present inventors have found that when a coloring matter is added to a bioluminescence reaction system, luminescence with desired color tone can be obtained.
Further, this luminescent plaything has the disadvantage that it is poor in stability to light and oxidation, and its luminescence stability is lost in a short period.
As a result of the intensive study under these circumstances, the present inventors have found that even if at least 2 components out of components necessary for bioluminescence are integrally mixed, the inhibition of luminescence does not occur, and this mixture is put in a light- and gas-impermeable container, and optionally at least one matter selected from the group consisting of an antioxidant, a desiccant and an inert gas is further introduced into the container which is in turn closed tightly, whereby it is possible to prevent a reduction in luminescence stability, and this tightly closed container is stored under forcible cooling whereby it is possible to prevent a reduction in luminescence stability.
The present invention provides a luminescent plaything comprising a composition containing 3 components of luciferin, luciferase and ATP as bioluminescent components. These 3 components is substantially free of a metal salt.
The present invention also relates to a luminescent plaything comprising a combination of environmental water and a composition containing 3 components of luciferin, luciferase and ATP, the environmental water and the composition being not simultaneously mixed in a liquid state. Here, the environmental water includes seawater, lake and marsh water, river water, ground water, tap water, and mineral drinking water.
The present invention further provides a luminescent plaything comprising a composition containing 4 components of luciferin, luciferase, ATP and a metal salt as bioluminescent components, the 4 components being not simultaneously mixed in a liquid state.
The present invention provides a luminescent plaything comprising a composition containing 5 components of luciferin, luciferase, ATP, a metal salt and a buffer agent as bioluminescent components, the 4 components of luciferin, luciferase, ATP and a metal salt being not simultaneously mixed in a liquid state.
The contents of luciferin, luciferase, ATP and a metal salt are 0.01 mM or more, 0.015 mg/ml or more, 0.002 mM or more, and 0.02 mM or more, respectively.
The present invention provides a luminescent plaything comprising a composition containing pyruvate orthophosphate dikinase, phosphoenolpyruvic acid, pyrophosphoric acid, luciferin, luciferase, ATP and a metal salt as luminescent components, the 4 components of luciferin, luciferase, ATP and a metal salt being not simultaneously mixed in a liquid state.
The present invention provides a luminescent plaything comprising a composition containing 4 components of luciferin, luciferase, ATP and a metal salt as luminescent components, the 4 components being not simultaneously mixed in a liquid state, at least one of the 4 components being a granulated material.
The present invention provides a luminescent plaything comprising a composition containing 4 components of luciferin, luciferase, ATP and a metal salt as luminescent components, the 4 components being not simultaneously mixed in a liquid state and being integrally granulated. The granulated material may be in the form of granules, capsules, tablets, pellets, sheets, flakes or beads.
The present invention provides a luminescent plaything comprising luminescent components in which at least one of 4 components of luciferin, luciferase, ATP and a metal salt is absorbed into or adhered to a carrier, and the absorbed or adhered component emits a light via water containing the remaining components or via water. The exemplary carrier includes fabrics, nonwoven fabrics, fibers, woods, papers, sponges, mesh structures and dried flowers.
The present invention provides a luminescent plaything wherein at least one or all of the components necessary for luminescence, after optionally granulated or after adsorbed into or adhered to a carrier, are covered on the carrier surface with a water-soluble substance.
The present invention provides a method for generating bioluminescence which comprises adding a luminescence-aiding material consisting of an arbitrary shape of a water-insoluble body to a bioluminescent liquid. The bioluminescence-aiding material is, for example, one having a gloss surface. The bioluminescent liquid may comprise a composition containing 4 components of luciferin, luciferase, ATP and a metal salt, the 4 components being not simultaneously mixed in a liquid state.
The present invention provides a luminescent plaything comprising a combination of bioluminescent components and a coloring matter.
The present invention provides a method for generating bioluminescence which comprises adding a coloring matter to the bioluminescent components in order to change a color tone of luminescence.
Examples of the luminescent plaything include candles, table lights, pen lights, illumination, illuminating playthings for camps, illuminating playthings for night fishings, fish-gathering lamps, safety candles, neon lights, lights on ice, luminescent inks, luminescent pens, luminescent coatings, or luminescent writing implements. The lights on ice make use of 4 components of luciferin, luciferase, ATP and a metal salt as components in the form of powder, and these components are dissolved in divided portions or in one portion in the surface water on ice thereby emitting a light. The luminescent writing implements comprise a composition containing 4 components of luciferin, luciferase, ATP and a metal salt as bioluminescent components, the 4 components being not simultaneously mixed in a liquid state, wherein the 4 components are divided into a group of a luminescent base ink containing at least one of the components and a group of an luminescent ink ancillary material containing the other components, and one of the groups is fixed by adhesion or absorption to a carrier, and the other group is maintained in an ink accommodating section in a writing implement so that the writing implement can be used to write on the surface of the carrier thereby emitting a light.
The present invention provides a method of storing a bioluminescent composition wherein, of components necessary for bioluminescence, at least 2 components are put in a gas- and light-impermeable container and closed tightly.
The present invention provides a method of storing a bioluminescent composition wherein, of components necessary for bioluminescence, at least 2 components are put in a gas- and light-impermeable container, and simultaneously at least one matter selected from the group consisting of an antioxidant, a desiccant and an inert gas is added thereto, and then the container is closed tightly.
The present invention provides a method of storing a bioluminescent composition wherein, of components necessary for bioluminescence, at least 2 components are put in a gas- and light-impermeable container and closed tightly, and the container is stored under forcible cooling.
Here, the components necessary for bioluminescence may comprise a composition containing 4 components of luciferin, luciferase, ATP and a metal salt, the 4 components being not simultaneously mixed in a liquid state. Further, the light- and gas-impermeable container is a plastic bag container made of a film having a metal film vapor-deposited on a synthetic resin film, a metal film, or a film having a metal film laminated on the synthetic resin film, or a hollow container made of synthetic resin or glass covered thereon with a light-impermeable film, coated with a light-impermeable coating, or made of brown or black materials.
Hereinafter, the present invention will be described in detail.
First, the luciferin used in the present invention refers to luciferin, luciferin derivatives and luciferin precursors.
The luciferin derivatives include, for example, D-luciferin-O-sulfate, D-luciferin methyl ester, D-luciferin-O-phosphate, D-luciferin-L-phenylalanine, D-luciferin-L-Nxcex1-arginine, and the like.
The luciferin derivatives include, for example, 2-cyano-6-methoxybenzothiazole, D-cysteine, and the like.
The luciferase includes, for example, luciferases derived from Coleoptera such as Luciola cruciata, Luciola lateralis, Luciola mingrelica, Lampyria nactiluca, Photinus pryalis, Photuris pennsylvanica, Hotaria parvula, Pyrocoelia miyako, and Pyrophorus plagiophthalamus, or these luciferases or mutants thereof produced by genetic recombination as well as other luciferases derived from non-Coleoptera.
ATP means ATP, its salts, and an ATP-forming reaction system.
The ATP-forming reaction system includes, for example, a method of forming ATP from phosphoenolpyruvic acid and ADP in the presence of pyruvate kinase as a catalyst, a method of forming ATP from creatine phosphoric acid and ADP in the presence of creatine kinase as a catalyst, and other methods.
The metal salt means divalent metal salts, for example, magnesium salts such as magnesium sulfate, magnesium chloride, and the like, manganese salts such as manganese sulfate, manganese chloride, and the like.
In the present invention, a buffer or an enzyme stabilizer can also be contained in addition to the components used.
The buffer is suitably used for maintaining the luminescence reaction solution in the optimum pH range for luciferase, and includes, for example, glycine-NaOH buffer, HEPES buffer, Tris buffer, etc. The addition of such a buffer (or buffer solution) results in significant extension of luminescence.
The exemplary enzyme stabilizer includes sugars, such as sucrose, and glycerol.
By adding the enzyme stabilizer, the effect of significant extension of luminescence is attained.
In the luminescent plaything according to the present invention, it is important that the luminescent plaything comprises a combination of environmental water and a composition containing 3 components of luciferin, luciferase and ATP substantially free of a metal salt, the 3 components and environmental water being not simultaneously mixed in a liquid state. If the 3 components and environmental water are mixed simultaneously in a liquid state, luminescence reaction is rapidly initiated, and after the luminescence reaction is finished, the plaything looses its value as a commodity.
In the present invention, the term xe2x80x9csubstantially free of a metal saltxe2x80x9d means that each of the 3 components does not contain a metal salt at all or that a metal salt, if any, hardly has the ability to promote luminescence. This can be achieved by preventing any contamination of metal salts when the 3 components are prepared, or by addition of a chelating agent, if the contamination can not be prevented, resulting in inhibition of a luminescence reaction caused by a metal salt.
The luminescent plaything lacking at least one of the 3 components fails to occur a luminescence reaction and, in this case, it is impossible to utilize as a luminescent plaything. Further, even the combination of the 3 components and water fails substantially to occur a fluorescence reaction if the water is pure water (or distilled water) or deionized water free of metal salts rather than environmental water, so it cannot be used. Thus, pure water or deionized water cannot be utilized in the luminescent plaything of the present invention, but the pure or deionized water in which a metal salt(s) (magnesium salt, manganese salt, etc.) is dissolved can rapidly initiate a luminescence reaction and so this characteristic can be utilized in luminescent playthings.
The luminescent plaything comprising a combination of the 3 components of luciferin, luciferase and ATP substantially free of a metal salt can initiate luminescence when mixed with environmental water, particularly with seawater, and the luminescent plaything which, by virtue of this characteristic, is preferable as an illuminating plaything for night fishing or as a fish-gathering lamp can be obtained.
That is, the illuminating plaything for night fishing is a fishing tackle such as a fishing line, a hook (including a fly), a sinker, a float or a fishing rod, which brightly illuminates in water as a whole or topically. The fish-gathering lamp is used to improve fishing efficiency by use of the property (i.e., taxis) of fish gathering toward light, and in this case the luminescent plaything itself is used in water.
For use in water, there are the following methods: 1) the luminescent plaything of the present invention in the form of liquid, solid or powder, directly or after encapsulated in a water-permeable or water-impermeable packaging bag, is introduced into or embedded in a bait (e.g., fly, natural, synthetic or semi-synthetic bait, or chum) or mixed with a bait for aquatic animals (e.g., mollusks such as cuttlefish, octopus and lobster, and fishes) living in sea, rivers, lakes andmarshes, or applied onto (adhered to) the surface of the bait, whereby the bait itself emits light; and 2) the luminescent plaything is kept near the bait or a hook, whereby the vicinity of the bait and hook is kept bright.
It is also important that the luminescent plaything of the present invention comprises the 4 components of luciferin, luciferase, ATP and a metal salt which are not simultaneously contained in a liquid state. If the 4 components are simultaneously contained in a liquid state, then a luminescence reaction is rapidly initiated, and after the luminescence reaction is finished, the plaything looses its value as a commodity.
However, if the 4 components are not in a liquid state, i.e., if they are in the form of dry powder, no luminescence reaction occur even if they are contained simultaneously, and in this case, use as a luminescent plaything is possible.
In this case, a truly magic luminescent plaything which, upon being merely mixed with water or a buffer, initiates a luminescence reaction can be provided.
Further, the luminescent plaything of the present invention is a luminescent plaything comprising a combination containing 4 components of luciferin, luciferase, ATP and a metal salt, the 4 components being not simultaneously mixed in a liquid state, wherein the 4 components are divided into a group of a luminescent base ink containing at least one of the 4 components and a group of an luminescent ink ancillary material containing the other components, and one of the groups is fixed by adhesion or absorption to a carrier (e.g., paper, woven fabric, nonwoven fabric, wood, etc.), and the other group is maintained in an ink accommodating section in a writing implement so that the writing implement can be used to write on the surface of the carrier thereby emitting a light, whereby one can draw luminescent letters, luminescent figures, luminescent drawings, etc.
It is also important that the 4 components of luciferin, luciferase, ATP and a metal salt have the final concentrations of 0.01 mM or more, 0.015 mg/ml or more, 0.002 mM or more, and 0.02 mM or more, respectively. If the contents of the respective components are lower than the above values, luminescence which is satisfactory when observed with the naked eye and worthy of appreciation cannot be obtained.
The concentrations of the 4 components, and the preferable concentration ranges of a buffer and an enzyme stabilizer, if used, are as follows (shown as final concentrations):
(1) Luciferin:
0.01 mM or more, preferably 0.03 to 50 mM, most preferably 0.10 to 12 mM.
(2) Luciferase:
0.015 mg/ml or more, preferably 0.05 to 20.0 mg/ml, most preferably 0.15 to 5 mg/ml.
(3) ATP:
0.002 mM or more, preferably 0.02 to 50 mM, most preferably 0.2 to 20 mM.
(4) Magnesium Sulfate (metal salt):
0.02 mM or more, preferably 0.1 to 300 mM, most preferably 0.5 to 100 mM.
(5) pH of Buffer:
pH 5.0 to 11.0, preferably 6.0 to 10.0, most preferably 7.0 to 9.0. e.g., Glycine-NaOH Buffer (pH 7.5):
0.01 mM or more, preferably 1 mM to 2.0 M, most preferably 0.1 to 1.0 M.
(6) Sucrose (enzyme stabilizer):
0.1 mM or more, preferably 1 to 300 mM, most preferably 10 to 50 mM.
It is also important that the luminescent plaything of the present invention comprises a combination containing pyruvate orthophosphate dikinase, phosphoenolpyruvic acid, pyrophosphoric acid, luciferin, luciferase, ATP and a metal salt, the 4 components of luciferin, luciferase, ATP and a metal salt being not simultaneously mixed in a liquid state.
According to this invention, said combination is mixed via water whereby a luminescent plaything having high luminescence stability can be obtained.
The present invention is characterized in that as shown in the following reaction system, the reaction shown in the broken line (A) in which pyruvate orthophosphate dikinase is allowed to act on AMP, pyrophosphoric acid, phosphoenolpyruvic acid and magnesium ions to convert them into ATP, pyruvic acid and phosphoric acid is combined with the reaction shown in the broken line (B) in which luciferase is allowed to act on ATP, luciferin, dissolved oxygen and magnesium ion to generate AMP, pyrophosphoric acid, oxyluciferin, carbon dioxide gas and light. 
In the reaction system, ATP is consumed in the reaction (B) with occurrence of luminescence thereby forming AMP and pyrophosphoric acid, which in turn contribute the regeneration of ATP in the reaction (A). The regenerated ATP is subjected again to the reaction (B) where the ATP is consumed with occurrence of luminescence.
Thereafter, these two reactions proceed simultaneously, and luminescence is kept at a constant high level for at least 10 minutes without decay of luminescence.
The pyruvate orthophosphate dikinase used in the present invention is an enzyme which catalyzes the reaction of generating ATP, pyruvic acid and phosphoric acid by acting on AMP, phosphoenolpyruvic acid and pyrophosphoric acid in the presence of a magnesium ion and also catalyzes its reverse reaction. Physicochemical properties of the enzyme, as well as production processes thereof, have already been known, and the enzyme itself is readily available.
The enzymes, derived from microorganisms and plants are known.
The microorganism-derived enzymes includes, for example, enzymes produced by microorganisms such as Propionibacterium shermanii [Biochemistry 10, 721-729 (1971)], Acetobacter xylinum [Journal of Bacteriology (1970)], Bacteroides symbiosus [Methods in Enzymology 42, 199-212 (1975)], and microorganisms belonging to the genus Microbispora (e.g. Microbispora thermorosea IFO14047).
Further, the plant-derived enzymes include, for example, enzymes derived from corn leaves [Biochemistry 12, 2862-2867 (1973)] and sugarcane leaves [The Biochemical Journal 114, 117-125 (1969)].
One example of the process for producing pyruvate orthophosphate dikinase by a microorganism is set forth below:
50 ml of a medium (pH 7.0) containing 0.2% yeast extract, 0.2% casamino acid, 0.001% ferrous sulfate, 0.05% potassium chloride, 0.1% dipotassium phosphate, 0.05% magnesium sulfate, and 0.3% lactic acid is put in an Erlenmmeyer flask (500 ml volume) and then sterilized at 121xc2x0 C. for 15 minutes.
Microbispora thermoroaes IFO14047 is inoculated into this medium and cultured at 45xc2x0 C. overnight under shaking to give a culture.
50 ml of the obtained culture is inoculated into a 5-L Erlenmmeyer flask containing 1 L medium having the same composition as above and cultured overnight to give a culture.
500 ml of the resulting culture is inoculated into each of two 30-L jar fermentors each containing 20 L medium with the same composition as described above and then cultured under shaking at an aeration rate of 20 L/min. at a stirring rate of 300 r.p.m. at 45xc2x0 C. for 24 hours.
At the end of the cultivation, the microorganism is harvested from 40 L of the culture through Microsa (Asahi Chemical Industry Co., Ltd.). 20 mM HEPES buffer (pH 7.5) containing 5 mM EDTA, 1 mM MgSO4 and 1 mM DTT (referred to hereinafter as buffer A) is added to part (200 g) of the microorganism, which is then suspended sufficiently to give a 700 ml suspension.
Purificaiton of pyruvate Orthophosphate Dikinase:
700 ml of the above-obtained microbial suspension is purified in the following steps:
step 1
(Preparation of Crude Enzyme Solution)
8.75 g of lysozyme (Nagase Seikagaku Kogyo K. K.) is added to 700 ml of the above microbial suspension, and the suspension is left for 2 hours at room temperature under gentle stirring. Then, 23.1 g of diammonium phosphate is added thereto, followed by stirring at room temperature for 2 hours to disrupt the microorganism.
The resulting liquid is centrifuged at 7000 r.p.m. for 15 minutes, and the supernatant is thus collected to give a 620 ml liquid.
step 2
(First QAE-Sephadex Chromatography)
Sulfate ammonium is dissolved at the concentration of 2 g/100 ml in 620 ml of the liquid of step 1, and this solution is passed through about 700 ml QAE-Sephadex resin (Pharmacia) previously equilibrated with buffer A (pH 7.5) containing 0.15 M ammonium sulfate whereby the enzyme is adsorbed onto the resin. It is washed with buffer A (pH 7.5) containing 0.15 M ammonium sulfate to remove unnecessary proteins, and the enzyme is eluted with buffer A (pH 7.5) containing 0.6 M ammonium sulfate.
step 3
(Second QAE-Sephadex Chromatography)
The above eluate is concentrated in a hollow fiber ultrafiltration unit (PAN 13-DX, Asahi Medical Co., Ltd.) and then applied to a column (6 cm xc3x8xc3x9715 cm) packed with QAE-Sephadex previously equilibrated with buffer A (pH 7.5) whereby the enzyme is adsorbed on the resin. Then, the column is washed with buffer A (pH 7.5) containing 0.15 M ammonium sulfate to remove unnecessary proteins, and the enzyme is subsequently eluted with 3L of a buffer with a gradient from buffer A (pH 7.5) containing 0.15 M ammonium sulfate to buffer A (pH 7.5) containing 0.8 M ammonium sulfate.
step 4
(Butyl Toyopearl Chromatography)
Following recovery of the above active fraction, sulfate ammonium is added thereto to a concentration of 1 M. The resultant is applied to a column (4.5 cm xc3x8xc3x9715 cm) packed with Butyl Toypearl (Tosoh corporation) previously equilibrated with buffer A (pH 7.5) containing 1 M sulfate ammonium whereby the enzyme is adsorbed onto the resin.
The protein is eluted with 1.2 L buffer A (pH 7.5) with a gradient from 1.0 M to 0 M ammonium sulfate in buffer A (pH 7.5) (1.2 L in total).
step 5
(Gel Filtration Chromatography)
The active fraction obtained in step 4 is concentrated to 2 ml using Amicon ultrafiltration membrane unit (cut off:
10,000), and 100 xcexcl aliquot is subjected to gel filtration by passing it through a TSK gel G3000 SW XL column (Tosoh Corporation) (0.76 cm ▭xc3x9730 cmxc3x972 columns) previously equilibrated with 20 mM HEPES buffer (pH 7.5) containing 0.3 M ammonium sulfate.
The whole of the enzyme is subjected to gel filtration, and the obtained active fraction is concentrated. The active fraction is subjected to the same columns and the eluted active fraction, 5.4 ml, was collected.
This fraction is an authentic sample of the present enzyme which is determined to be homogeneous by SDS-polyacrylamide gel electrophoresis, and the total protein is 6.65 mg, the total activity is 66.0 U, and the specific activity is 9.92 U/mg.
Method of Measuring the Titer of Pyruvate Orthophosphate Kinase:
(Method of Quantifying Formed ATP by a Luminescent Method)
180 xcexcl of 50 mM BIS-TRIS PROPANE buffer (pH 6. 8) containing 3 mM magnesium sulfate, 25 mM ammonium sulfate, 2 mM 2-mercaptoethanol, 2 mM pyrophosphoric acid, 2 mM phosphoenolpyruvic acid and 0.1 mM AMP is put in a microtube. After the temperature is equilibrated at 37xc2x0 C., 20 xcexcl of an enzyme solution having a suitable activity is added to the tube and, the mixture is incubated for 15 minutes, then boiled in boiling water for 3 minutes to terminate the reaction. This reaction mixture is diluted suitably, and 50 xcexcl aliquot thereof is put in a test tube, 50 xcexcl xe2x80x9cLucifer LUxe2x80x9d solution (Kikkoman Corporation) is added dropwise thereto, and the level of luminescence is measured.
Separately, a calibration curve in which ATP standard solutions with predetermined concentrations are used to examine the relationship with luminescence levels is prepared.
This graph is used, and the amount of the enzyme for producing 1 xcexcmol ATP per minute at 37xc2x0 C. is arbitrarily defined as 1 U.
In the present invention, it is also important that at least one of the 4 components in the form of powder is granulated whereby the duration of luminescence can be significantly increased as compared with either case where all the 4 components are aqueous solutions or powder.
For granulation of the 4 components of luciferin, luciferase, ATP and a metal salt, these can be granulated in a usual manner into granules, capsules, tablets, pellets (including bars), sheets, flakes or beads, directly or after an excipient, a binder etc. are added to each of the 4 components or to a combination of 2 or more components.
To make granules, granulation processes such as wet granulation process and dry granulation process can be used.
In the wet granulation process, about 5 to 30 parts by weight of water is added to 100 parts by weight of materials to be granulated (however, water cannot be added simultaneously to the 4 components, so the 4 components should not be contained simultaneously in the materials to be granulated), and these are uniformly moistened and granulated in a granulator.
In this case, a granulator such as extrusion granulator, sand granulator or fluidized bed granulator is preferably used.
The resulting granules are dried to a water content of about 0.1-5% at a temperature of 90xc2x0 C. or less by drying such as air drying or fluidized bed drying.
In general, the granules are preferably prepared such that a particle size in the range of about 0.2 to 2 mm is achieved.
Then, the granules are sieved by a shifter or screen having a specific opening.
To make beads, the materials are formed into spherical or nearly spherical beads having particle sizes with a narrow distribution in the range of 0.5 to 20 mm by amicrocapsule method or a CF (centrifugation) method or by means of a sphere shifter at high-speed revolution.
To make tablets, the materials or the granules obtained by the method as described above are formed into tablets. The shape of tablets may be an arbitrary shape such as circle-, star-, heart-, ball-, triangle-, or alphabet-shape., and generally the tablets are preferably circle-shaped with concave or convex surface, having a diameter (or the maximum length) of about 2 mm to 10 cm and a thickness of about 0.5 mm to 5 cm and weighing about 10 mg to 50 g, preferably 100 mg to 25 g, more preferably about 200 mg to 5 g.
To make pellets, it is preferable that the materials are extruded into pellets (including bars) with a diameter of 0.2 to 10 mm and a length of 2 mm to 30 cm in an extrusion granulator.
When formed into long and narrow bars, it is preferable that these can be used like a pencil and a lead in a mechanical pencil.
The excipient includes starch, dextrin, lactose, egg white, soybean protein, gelatin, etc., and the binder includes carboxymethyl cellulose, alginic acid, salts thereof, polyvinyl alcohol, polyacrylic acid, salts thereof, polymethacrylic acid, gum arabic, sucrose, polysaccharides, starch pastes, fats and oils, etc.
The means of granulating at least one of the 4 components includes a method in which the luciferin, luciferase, ATP and a metal salt are formed into particles in a usual manner by granulating themselves or their mixture (also referred to hereinafter as materials to be granulated) having other components added thereto (e.g., at least one matter selected from a buffer, an enzyme stabilizer, a binder, an excipient, an extender, a pigment and a perfume may be added in such a range as not to inhibit luminescence).
Specifically, mention may be made of a method in which the respective components of luciferin, luciferase, ATP and a metal salt are separately granulated to produce 2 kinds or more of particles, or are integrally granulated after other components are optionally added thereto.
According to the present invention, a luminescent plaything with a luminescence duration improved by bringing it in contact with water can be obtained.
It is also important in the present invention that, of the 4 components, at least one or all components are adsorbed or adhered to a carrier.
That is, it is important that the 4 components or a combination of 2 or more components are adsorbed or adhered to a carrier in a usual manner directly or after an excipient, a binder etc. are added thereto, followed by natural drying or lyophilization.
The carrier used herein is at least one matter selected from the group consisting of fabric, nonwoven fabric (including felt), (natural, synthetic, or glass) fiber, wood, paper, a sponge-like structure (e.g., rubber-like material having holes thereon like sponge, such as polyurethane sponge, urethane sponge, or spongy carrier for chromatography), a mesh structure (e.g., a shaped article or an embodied object made of a mesh film of synthetic resin), and a dried flower.
The excipient includes starch, dextrin, lactose, egg white, soybean protein, gelatin, etc.
The binder includes carboxymethyl cellulose, alginic acid, salts thereof, polyvinyl alcohol, polyacrylic acid, salts thereof, polymethacrylic acid, gumarabic, starchpaste, sucrose, polysaccharides, fats and oils, etc.
According to the present invention, the luminescent plaything can be produced in this manner in any shape such as planar shapes (cubic, square, regular square, rectangular, triangular, trapezoid, pentagonal, hexagonal, polygonal, and circular), three-dimensional shapes (e.g., prism, cubic, rectangular parallel-epiped, and cylindrical), spherical shapes, pyramidal shapes (e.g., conical, pentagonal, hexagonal, and polygonal), letter shapes (e.g., alphabets, katakana, hiragana, and numerals), determined shapes (e.g., heart, egg, bow, fan, turtle shell, wedge, stripe, and wave-shaped), logo marks, symbol marks, signs, embodied objects (e.g., shapes embodying animals and plants), gardening articles, daily commodities, writing materials, office supplies, electric appliances, toys (e.g., dolls, stuffed toys, swords, guns, and robots), musical instruments, vehicles, buildings, etc.
Further, it is important in the present invention that, of the components necessary for bioluminescence, at least one or all components, after optionally granulated or after adsorbed or adhered to a carrier, are covered on the surface thereof with a water-soluble substance.
In the present invention, the means of granulating the components essential for luminescence includes a method in which the luciferin, luciferase, ATP and a metal salt are formed in a usual manner into granules, capsules, tablets, pellets (including bars), sheets, flakes or beads by granulating themselves or a combination of 2 or more components after other components are added thereto (e.g., at least one matter selected from a buffer, an enzyme stabilizer, a binder, an excipient, an extender, a pigment and a perfume may be added thereto in such a range as not to inhibit luminescence.).
In the method of allowing the 4 components of luciferin, luciferase, ATP and a metal salt necessary for bioluminescence to be absorbed, or adhered to a carrier in the present invention, the 4 components or a combination of 2 or more components are absorbed or adhered to the carrier in a usual manner directly or after an excipient, a binder etc. are added thereto, followed by natural drying or lyophilization.
To cover the surface of at least one or all of the components necessary for bioluminescence with a water-soluble substance, the components are immersed in (or brought into contact with) an aqueous solution (or a colloidal solution) of the water-soluble substance, followed by lyophilization, or alternatively the surface of the components is sprayed with the water-soluble substance and then slightly scattered with water whereby the substance is swollen and dissolved in the form of colloid, followed by lyophilization.
The surface of the components can thereby coated with the water-soluble substance.
The concentration of the water-soluble substance in an aqueous solution is 0.1 to 10 (W/W) %, preferably 0.5 to 3%.
The water-soluble substance used herein is one or more matters selected from carboxymethyl cellulose, carboxyethyl cellulose, starch, dextrin, lactose, eggwhite, soybeanprotein, gelatin, alginic acid and salts thereof, polyvinyl alcohol, polyacrylic acid and salts thereof, polymethacrylic acid, gum arabic, starch paste, sucrose and polysaccharides.
Among these, carboxymethyl cellulose, carboxyethyl cellulose and gelatin are particularly preferable.
The water-soluble substance, when brought into contact with a small amount of water, is swollen with moisture to form colloid, and by suitably controlling the amount of water, a coating having a different thickness can be formed on the surface of the luminescence components.
If the coating having a different thickness is formed on the surface of the luminescent components, the luminescent plaything can topically emit a light when it has reached an objective depth (or a point) in seawater, so it can be used preferably as a luminescent bait for fishing.
As the luminescent bait for fishing, the luminescent plaything can be used as such, but preferably it is used after mixed with a known bait for fishing.
The bait for fishing includes alive fishes, natural baits such as cut fishes, shrimps, cuttlefishes, krill, annelids etc., or synthetic baits such as mixtures of fish ground meats or extracts and starch, potatoes etc. or mixtures of fish meals or fish and shellfish extracts and a gelling agent for food.
Further, it is important in the present invention that the above-described bioluminescent fluid is provided therein with a luminescence-aiding material consisting of an arbitrarily shaped water-insoluble body.
The water-insoluble material used in the present invention may be made of any arbitrary water-insoluble material. For example, mention is made of glass, crystal glass, synthetic resin (plastic), metal, wood, paper, rubber, stone, ceramic ware, natural flowers and trees, minerals, wax etc.
Further, the water-insoluble body includes (1) ball (or marbles), beads, polyhedron body (e.g. mirror ball), ring, rock ice (rock-shaped ice-like plastic or acrylic materials), fiber body, (2) film, (3) metal foil such as gold foil, aluminum foil etc., (4) natural flowers and trees, alive flowers and dried flowers, (5) articles (e.g. artificial flowers) embodying natural flowers and woods, animals, dolls, ornaments, toys, and small equipment, (6) precious stones such as jewels, ores, natural stones, imitation jewels etc., (7) candles, and (8) accessories such as bracelets, rings, earrings, pendants etc.
These water-insoluble bodies should have such a size that they can be provided in the bioluminescent liquid, but their shape, color and transparency may be arbitrary.
Specifically, mention can be made of those which are colorless and transparent, colored and transparent, colorless and semitransparent, colored and semitransparent, colorless and opaque, and colored and opaque.
If water-insoluble bodies with gloss on the surface, e.g., a plurality of glass marbles, rock ices, jammed bent plastic film, metal foil etc., can be preferably used in the present invention to provide a luminescent liquid with luminescence emitted irregularly thereby increasing the glitter and further increasing the beauty.
The method of providing the water-insoluble body in the luminescent liquid includes a method of floating it on the surface of the luminescent liquid or a method of sinking a part or the whole of it in the luminescent liquid.
According to the present invention, the liquid emitting bioluminescence can be provided therein with water-insoluble bodies such as marbles, rock ices, plastic film or metal foil, whereby there can be provided a luminescence-aiding material having the function of emitting the luminescence irregularly thereby increasing the glitter and increasing the beauty as well as the luminescent method of using the luminescence-aiding material. Further, by providing the water-insoluble material therein, there can be provided a luminescent plaything which is attractive even using a small amount of the luminescent liquid.
Further, it is important in the present invention that a coloring matter is added to the bioluminescence reaction system whereby emission having a desired tone of color is obtained.
The coloring matter used herein includes pigments, coloring agents (containing luminescent coloring agents) and paints. The pigments include edible red No. 3 (dark pink), red edible red No. 102 (dark red), edible yellow No. 5 (yellow), edible green No. 3 (bluish green), edible blue No. 1 (blue), edible blue No. 2 (ultramarine), xcex2-carotin pigment (bright golden yellow), paprika pigment (cinnabar), paprika pigment (orange), gardenia yellow pigment (lemon yellow), annatto pigment (dark orange), annatto pigment (orange), berry pigment (red wine color), cacao pigment (dark brown), kaoliang pigment (pale brown), shisonin pigment (violet), chotinyl pigment (rose wine color), copper chlorophylline sodium (green), Monascus pigment (reddish violet), Monascus yellow pigment (luminescent lemon yellow), gardenia blue pigment (dark ultramarine), BV14 MAGENTA pdr (red, from Daimatsu Sangyo K. K.), Suminol Milling Green G (green, from Sumitomo Chemical Co., Ltd.) etc., and the paints include watercolors. These are used preferably at concentrations of 0.00001 to 3.0 (W/V) %, more preferably 0.001 to 1.0 (w/v) %.
Further, it is also important in the present invention that of the bioluminescent components necessary for bioluminescence, at least 2 components are introduced into a light- and gas-impermeable container and closed tightly.
Further, it is also important that the container is stored under forcible cooling.
In the present invention, at least 2 components of the components necessary for bioluminescence are mixed.
That is, at least 2 components, preferably 3 components, and more preferably all the components (excluding the case where they are in a liquid state) are mixed.
It is important that at least 2 components are mixed (integrated into one body). If the components necessary for luminescence are used (individually), that is, if each component is packaged, it is the trouble that each component should be packaged and handled for use, and the operation of mixing the respective components at a suitable mixing ratio is troublesome.
However, if at least 2 components of the components necessary for bioluminescence are mixed (even if the 2 components are mixed, the inhibition of luminescence does not occur as described above), it is easier to mix the components, or if all the components (as the dried matter) are mixed (where the inhibition of luminescence does not occur as described above), the mixture can achieve the object (i.e. generation of fluorescence) by merely opening its container and then mixing it with water or a buffer solution.
The light- and gas-impermeable container used in the present invention includes film-shaped bag containers made of (1) a single film consisting of polyethylene, polypropylene etc., or a film having a metal such as aluminum vapor-deposited on a film of synthetic resin such as polyester, oriented nylon or cellophane with a polyolefin thermal adhesion resin laminated thereon, (2) a metal film such as aluminum foil etc., (3) a film having a metal film laminated on the synthetic resin film, and hollow containers (plastic blown bottles, glass bottles etc.) such as (4) a hollow container covered thereon with a light-impermeable film, (5) a hollow container coated with a light-impermeable coating and (6) a hollow container made of brown or black materials.
In the present invention, it is important to use the light- and gas-impermeable container, and when a light- and/or gas-permeable container is used, it is not possible to prevent reduction of luminescence stability.
When the bioluminescent composition is introduced into the light- and gas-impermeable container and closed tightly, an antioxidant, a desiccant or an inert gas is preferably added thereto to prevent the reduction of luminescence stability.
The antioxidant may be any arbitrary material by which the reduction of luminescence performance by air oxidation can be prevented, and examples thereof include commercial oxygen absorbers.
The desiccant may be any arbitrary material by which the reduction of luminescence performance by moisture absorption can be prevented, and examples thereof include silica gel, calcium chloride, aluminum oxide, dry soda etc.
The inert gas includes nitrogen, hydrogen, argon, carbon dioxide etc. The air in a head space (gaseous phase) in the container is replaced by the inert gas whereby the reduction of luminescence stability by the oxidation of the bioluminescent composition can be prevented.
Further, it is also important in the present invention that the components necessary for bioluminescence are stored under forcible cooling, and if the components are stored at room temperature, there is the problem of inevitable reduction of luminescence stability.
For forcible cooling, the components as such or after frozen in a refrigerator are stored (1) under cooling in a freezer or (2) with a cooling medium in a container made of a thermal insulation material.
The cooling medium may be an arbitrary medium with which the temperature in the container containing the bioluminescent composition can be kept at a low temperature, and examples thereof include dry ice, crushed ice or cooling materials (e.g. synthetic CMC, a gel cooling agent etc.)
If the bioluminescent composition is liquid, the liquid is frozen in a freezing compartment so that the composition can also act as a cooling medium.
The thermal insulation material used for producing the container includes a single material such as foamed styrol, foamed urethane, foamed polyethylene or rigid urethane foam, or a combination thereof with other materials such as aluminum, stainless steel, titanium, polyethylene, polypropylene, avinyl chloride steel plate, quilting etc.
The components are stored preferably under forcible cooling in this manner in order to maintain their luminescence stability.